The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis.

Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid) is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and branched-chain α-ketoacid levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here, we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show that BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly sixfold less potent than the prototypical uncoupler 2,4-dinitrophenol and phenocopies 2,4-dinitrophenol in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest that the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

Structure-guided engineering of branched-chain α-keto acid decarboxylase for improved 1,2,4-butanetriol production by in vitro synthetic enzymatic biosystem.

Efficient synthetic routes for biomanufacturing chemicals often require the overcoming of pathway bottlenecks by tailoring enzymes to improve the catalytic efficiency or even implement non-native activities. 1,2,4-butanetriol (BTO), a valuable commodity chemical, is currently biosynthesized from D-xylose via a four-enzyme reaction cascade, with the ThDP-dependent α-keto acid decarboxylase (KdcA) identified as the potential bottleneck. Here, to further enhance the catalytic activity of KdcA toward the non-native substrate α-keto-3-deoxy-xylonate (KDX), in silico screening and structure-guided evolution were performed. The best mutants, S286L/G402P and V461K, exhibited a 1.8- and 2.5-fold higher enzymatic activity in the conversion of KDX to 3,4-dihydroxybutanal when compared to KdcA, respectively. MD simulations revealed that the two sets of mutations reshaped the substrate binding pocket, thereby increasing the binding affinity for KDX and promoting interactions between KDX and cofactor ThDP. Then, when the V461K mutant instead of wild type KdcA was integrated into the enzyme cascade, a 1.9-fold increase in BTO titer was observed. After optimization of the reaction conditions, the enzyme cocktail contained V461K converted 60 g/L D-xylose to 22.1 g/L BTO with a yield of 52.1 %. This work illustrated that protein engineering is a powerful tool for modifying the output of metabolic pathway.

Successful treatment of severe MSUD in Bckdhb-/- mice with neonatal AAV gene therapy.

Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

Reactive nitrogen species inhibit branched chain alpha-ketoacid dehydrogenase complex and impact muscle cell metabolism.

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex-RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases.

Branched-chain amino acids promote thrombocytopoiesis by activating mTOR signaling.

BACKGROUND: Megakaryocyte differentiation and platelet production disorders are the main causes of thrombocythemia and thrombocytopenia and lead to thrombosis or hemorrhage. Branched-chain amino acids (BCAAs) are essential nutrients that regulate important metabolic signals. BCAA administration could also increase platelet activation and promote the risk of thrombosis. OBJECTIVES: To unveil the role of BCAAs in thrombocytopoiesis. METHODS: BCAA-fed mice and megakaryocyte/platelet-specific branched-chain α-keto acid dehydrogenase E1α subunit-deficient mice were used to study the role of BCAAs in thrombocytopoiesis. RESULTS: In this study, we found that BCAA diet could facilitate megakaryocyte differentiation and platelet production. Meanwhile, megakaryocyte/platelet-specific branched-chain α-keto acid dehydrogenase E1α subunit-deficient mice developed thrombocythemia, which was mainly caused by the excessive differentiation of megakaryocytes and proplatelet biogenesis. Moreover, the use of BT2, the agonist of BCAA catabolism, could affect proplatelet formation (PPF) and megakaryocyte polyploidization, as well as ameliorating the thrombocythemia of BCAA-fed mice. CONCLUSION: We found that deficiency in BCAA catabolism led to the activation of p70S6K/mammalian target of rapamycin (mTOR) signaling, megakaryocyte over differentiation, and the acceleration of PPF. Activating BCAA metabolism with BT2 could inhibit mTOR signaling, reduce PPF, and ameliorate thrombocythemia in BCAA-fed mice. Therefore, this study reveals a novel role of BCAAs in megakaryocyte differentiation and platelet production, suggesting that targeting BCAA-mediated p70S6K/mTOR signaling may be a potential strategy for the treatment of thrombocytopenia or thrombocythemia.

Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism.

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. METHODS: Circulating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE; NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines and mouse primary hepatocytes were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA metabolism in the liver of obese mice and MSDC-0602K treatment of Zucker diabetic fatty (ZDF) rats. RESULTS: In patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, had decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades in vitro. BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2-/-) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo. Finally, while MSDC-0602K treatment improved glucose homeostasis and increased the concentrations of some BCAA metabolites in ZDF rats, it did not lower plasma BCAA concentrations. CONCLUSIONS: These data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the mTOR axis. However, the effects of MPCi on glucose homeostasis may be separable from its effects on BCAA concentrations.

Structural studies identify angiotensin II receptor blocker-like compounds as branched-chain ketoacid dehydrogenase kinase inhibitors.

The mammalian mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched-chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched-chain amino acids and branched-chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker class antihypertension drugs and angiotensin receptor blocker-like compounds were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and antihypertension.

PPM1K defects cause mild maple syrup urine disease: The second case in the literature.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by the insufficient catabolism of branched-chain amino acids. BCKDHA, BCKDHB, DBT, and DLD encode the subunits of the branched-chain α-ketoacid dehydrogenase complex, which is responsible for the catabolism of these amino acids. Biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT are characteristic of MSUD. In addition, a patient with a PPM1K defect was previously reported. PPM1K dephosphorylates and activates the enzyme complex. We report a patient with MSUD with mild findings and elevated BCAA levels carrying a novel homozygous start-loss variant in PPM1K. Our study offers further evidence that PPM1K variants cause mild MSUD.

Activation of Hepatic Branched-Chain α-Ketoacid Dehydrogenase Complex by Vitamin D Deficiency in Rats.

Branched-chain α-ketoacid dehydrogenase (BCKDH) complex is a rate-limiting enzyme in branched-chain amino acid catabolism and is subject to inactivation via phosphorylation by BCKDH kinase (BDK). In the present study, we examined the effects of vitamin D-deficiency on hepatic BCKDH and BDK activities in rats. Rats fed a vitamin D-deficient diet long-term showed a slight but significant decrease in plasma Ca concentration, which was associated with an elevation of BCKDH activity and a decrease in BDK activity. These results suggest that vitamin D deficiency promotes BCAA catabolism via BCKDH activation, which resulted from BDK suppression. It is proposed that Ca2+-dependent BDK inhibition by thiamine pyrophosphate may be involved in the BDK suppression.

BDK inhibition acts as a catabolic switch to mimic fasting and improve metabolism in mice.

OBJECTIVE: Branched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements. METHODS: The rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density lipoprotein receptor null mouse model (Ldlr-/-). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice. RESULTS: A rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr-/- mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-mediated gene expression in mice. CONCLUSIONS: These data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a "fed signal" to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a "fasting signal" to prolong the catabolic state.

Neonatal gene therapy achieves sustained disease rescue of maple syrup urine disease in mice.

Maple syrup urine disease (MSUD) is a rare recessively inherited metabolic disorder causing accumulation of branched chain amino acids leading to neonatal death, if untreated. Treatment for MSUD represents an unmet need because the current treatment with life-long low-protein diet is challenging to maintain, and despite treatment the risk of acute decompensations and neuropsychiatric symptoms remains. Here, based on significant liver contribution to the catabolism of the branched chain amino acid leucine, we develop a liver-directed adeno-associated virus (AAV8) gene therapy for MSUD. We establish and characterize the Bckdha (branched chain keto acid dehydrogenase a)-/- mouse that exhibits a lethal neonatal phenotype mimicking human MSUD. Animals were treated at P0 with intravenous human BCKDHA AAV8 vectors under the control of either a ubiquitous or a liver-specific promoter. BCKDHA gene transfer rescued the lethal phenotype. While the use of a ubiquitous promoter fully and sustainably rescued the disease (long-term survival, normal phenotype and correction of biochemical abnormalities), liver-specific expression of BCKDHA led to partial, though sustained rescue. Here we show efficacy of gene therapy for MSUD demonstrating its potential for clinical translation.

The Critical Role of the Branched Chain Amino Acids (BCAAs) Catabolism-Regulating Enzymes, Branched-Chain Aminotransferase (BCAT) and Branched-Chain α-Keto Acid Dehydrogenase (BCKD), in Human Pathophysiology.

Branched chain amino acids (BCAAs), leucine, isoleucine and valine, are essential amino acids widely studied for their crucial role in the regulation of protein synthesis mainly through the activation of the mTOR signaling pathway and their emerging recognition as players in the regulation of various physiological and metabolic processes, such as glucose homeostasis. BCAA supplementation is primarily used as a beneficial nutritional intervention in chronic liver and kidney disease as well as in muscle wasting disorders. However, downregulated/upregulated plasma BCAAs and their defective catabolism in various tissues, mainly due to altered enzymatic activity of the first two enzymes in their catabolic pathway, BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKD), have been investigated in many nutritional and disease states. The current review focused on the underlying mechanisms of altered BCAA catabolism and its contribution to the pathogenesis of a numerous pathological conditions such as diabetes, heart failure and cancer. In addition, we summarize findings that indicate that the recovery of the dysregulated BCAA catabolism may be associated with an improved outcome and the prevention of serious disease complications.

AICAR stimulates mitochondrial biogenesis and BCAA catabolic enzyme expression in C2C12 myotubes.

Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism. Insulin resistant populations often exhibit reduced expression of genes governing mitochondrial metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Interestingly, PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, the consistently observed increased circulating levels of BCAA in diabetics may be partially explained by reduced PGC-1α expression. Conversely, PGC-1α upregulation appears to increase BCAA catabolism. PGC-1α activity is regulated by 5'-AMP-activated protein kinase (AMPK), however, only limited experimental data exists on the effect of AMPK activation in the regulation of BCAA catabolism. The present report examined the effects of the commonly used AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the metabolism and expression of several related targets (including BCAA catabolic enzymes) of cultured myotubes. C2C12 myotubes were treated with AICAR at 1 mM for up to 24 h. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via qRT-PCR and western blot, respectively. AICAR treatment significantly increased mitochondrial content and peak mitochondrial capacity. AICAR treatment also increased AMPK activation and mRNA expression of several regulators of mitochondrial biogenesis but reduced glycolytic metabolism and mRNA expression of several glycolytic enzymes. Interestingly, branched-chain alpha-keto acid dehydrogenase a (BCKDHa) protein was significantly increased following AICAR-treatment suggesting increased overall BCAA catabolic capacity in AICAR-treated cells. Together, these experiments demonstrate AICAR/AMPK activation can upregulate BCAA catabolic machinery in a model of skeletal muscle.

BIX01294 inhibits EGFR signaling in EGFR-mutant lung adenocarcinoma cells through a BCKDHA-mediated reduction in the EGFR level.

BIX01294 (BIX), an inhibitor of the G9a histone methyltransferase, has been reported to have antitumor activity against a variety of cancers. However, the molecular mechanisms underlying its anticancer effects, particularly those against lung cancer, remain unclear. Here, we report that BIX induces apoptotic cell death in EGFR-mutant non-small cell lung cancer (NSCLC) cells but not in their wild-type counterparts. Treatment with BIX resulted in a significant reduction in the EGFR level and inhibition of EGFR signaling only in EGFR-mutant NSCLC cells, leading to apoptosis. BIX also inhibited mitochondrial metabolic function and decreased the cellular energy levels that are critical for maintaining the EGFR level. Furthermore, BIX transcriptionally downregulated the transcription of branched-chain α-keto acid dehydrogenase (BCKDHA), which is essential for fueling the tricarboxylic acid (TCA) cycle. Interestingly, this BCKDHA downregulation was due to inhibition of Jumanji-domain histone demethylases but not the G9a histone methyltransferase. We observed that KDM3A, a Jumonji histone demethylase, epigenetically regulates BCKDHA expression by binding to the BCKDHA gene promoter. BIX exposure also led to a significant decrease in the EGFR level, causing apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Taken together, our current data suggest that BIX triggers apoptosis only in EGFR-mutant NSCLC cells via inhibition of BCKDHA-mediated mitochondrial metabolic function.

Dihydrolipoamide dehydrogenase, pyruvate oxidation, and acetylation-dependent mechanisms intersecting drug iatrogenesis.

In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular homeostasis. The worldwide used antiepileptic drug valproic acid (VPA) may potentially induce teratogenicity or a mild to severe hepatic toxicity, where the underlying mechanisms are not completely understood. This work aims to clarify the mechanisms that intersect VPA-related iatrogenic effects to PDC-associated dihydrolipoamide dehydrogenase (DLD; E3) activity. DLD is also a key enzyme of α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, α-ketoadipate dehydrogenase, and the glycine decarboxylase complexes. The molecular effects of VPA will be reviewed underlining the data that sustain a potential interaction with DLD. The drug-associated effects on lipoic acid-related complexes activity may induce alterations on the flux of metabolites through tricarboxylic acid cycle, branched-chain amino acid oxidation, glycine metabolism and other cellular acetyl-CoA-connected reactions. The biotransformation of VPA involves its complete ß-oxidation in mitochondria causing an imbalance on energy homeostasis. The drug consequences as histone deacetylase inhibitor and thus gene expression modulator have also been recognized. The mitochondrial localization of PDC is unequivocal, but its presence and function in the nucleus were also demonstrated, generating acetyl-CoA, crucial for histone acetylation. Bridging metabolism and epigenetics, this review gathers the evidence of VPA-induced interference with DLD or PDC functions, mainly in animal and cellular models, and highlights the uncharted in human. The consequences of this interaction may have significant impact either in mitochondrial or in nuclear acetyl-CoA-dependent processes.

Association of BCAT2 and BCKDH polymorphisms with clinical, anthropometric and biochemical parameters in young adults.

BACKGROUND AND AIM: Circulating amino acids are modified by sex, body mass index (BMI) and insulin resistance (IR). However, whether the presence of genetic variants in branched-chain amino acid (BCAA) catabolic enzymes modifies circulating amino acids is still unknown. Thus, we determined the frequency of two genetic variants, one in the branched-chain aminotransferase 2 (BCAT2) gene (rs11548193), and one in the branched-chain ketoacid dehydrogenase (BCKDH) gene (rs45500792), and elucidated their impact on circulating amino acid levels together with clinical, anthropometric and biochemical parameters. METHODS AND RESULTS: We performed a cross-sectional comparative study in which we recruited 1612 young adults (749 women and 863 men) aged 19.7 ± 2.1 years and with a BMI of 24.9 ± 4.7 kg/m2. Participants underwent clinical evaluation and provided blood samples for DNA extraction and biochemical analysis. The single nucleotide polymorphisms (SNPs) were determined by allelic discrimination using real-time polymerase chain reaction (PCR). The frequencies of the less common alleles were 15.2 % for BCAT2 and 9.83 % for BCKDH. The subjects with either the BCAT2 or BCKDH SNPs displayed no differences in the evaluated parameters compared with subjects homozygotes for the most common allele at each SNP. However, subjects with both SNPs had higher body weight, BMI, blood pressure, glucose, and circulating levels of aspartate, isoleucine, methionine, and proline than the subjects homozygotes for the most common allele (P < 0.05, One-way ANOVA). CONCLUSION: Our findings suggest that the joint presence of both the BCAT2 rs11548193 and BCKDH rs45500792 SNPs induces metabolic alterations that are not observed in subjects without either SNP.

Genetic analysis by targeted next-generation sequencing and novel variation identification of maple syrup urine disease in Chinese Han population.

Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder that affects the degradation of branched chain amino acids (BCAAs). Only a few cases of MSUD have been documented in Mainland China. In this report, 8 patients (4 females and 4 males) with MSUD from 8 unrelated Chinese Han families were diagnosed at the age of 6 days to 4 months. All the coding regions and exon/intron boundaries of BCKDHA, BCDKHB, DBT and DLD genes were analyzed by targeted NGS in the 8 MSUD pedigrees. Targeted NGS revealed 2 pedigrees with MSUD Ia, 5 pedigrees with Ib, 1 pedigree with MSUD II. Totally, 13 variants were detected, including 2 variants (p.Ala216Val and p.Gly281Arg) in BCKDHA gene, 10 variants (p.Gly95Ala, p.Ser171Pro, p.Phe175Leu, p.Arg183Trp, p.Lys222Thr, p.Arg285Ter, p.Arg111Ter, p.S184Pfs*46, p.Arg170Cys, p.I160Ffs*25) in BCKDHB gene, 1 variant (p.Arg431Ter) in DBT gene. In addition, 4 previously unidentified variants (p.Gly281Arg in BCKDHA gene, p.Ser171Pro, p.Gly95Ala and p.Lys222Thr in BCKDHB gene) were identified. NGS plus Sanger sequencing detection is effective and accurate for gene diagnosis. Computational structural modeling indicated that these novel variations probably affect structural stability and considered as likely pathogenic variants.

Superior production of heavy pamamycin derivatives using a bkdR deletion mutant of Streptomyces albus J1074/R2.

BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

Insulin action, type 2 diabetes, and branched-chain amino acids: A two-way street.

BACKGROUND: A strong association of obesity and insulin resistance with increased circulating levels of branched-chain and aromatic amino acids and decreased glycine levels has been recognized in human subjects for decades. SCOPE OF REVIEW: More recently, human metabolomics and genetic studies have confirmed and expanded upon these observations, accompanied by a surge in preclinical studies that have identified mechanisms involved in the perturbation of amino acid homeostasis- how these events are connected to dysregulated glucose and lipid metabolism, and how elevations in branched-chain amino acids (BCAA) may participate in the development of insulin resistance, type 2 diabetes (T2D), and other cardiometabolic diseases and conditions. MAJOR CONCLUSIONS: In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding BCAA-restricted diet or by the activation of the rate-limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH), in rodent models of obesity have clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in short-term studies in human T2D subjects. Feeding of rats with diets enriched in sucrose or fructose result in the induction of the ChREBP transcription factor in the liver to increase expression of the BCKDH kinase (BDK) and suppress the expression of its phosphatase (PPM1K) resulting in the inactivation of BCKDH and activation of the key lipogenic enzyme ATP-citrate lyase (ACLY). These and other emergent links between BCAA, glucose, and lipid metabolism motivate ongoing studies of possible causal actions of BCAA and related metabolites in the development of cardiometabolic diseases.

Whole-body metabolic fate of branched-chain amino acids.

Oxidation of branched-chain amino acids (BCAAs) is tightly regulated in mammals. We review here the distribution and regulation of whole-body BCAA oxidation. Phosphorylation and dephosphorylation of the rate-limiting enzyme, branched-chain α-ketoacid dehydrogenase complex directly regulates BCAA oxidation, and various other indirect mechanisms of regulation also exist. Most tissues throughout the body are capable of BCAA oxidation, and the flux of oxidative BCAA disposal in each tissue is influenced by three key factors: 1. tissue-specific preference for BCAA oxidation relative to other fuels, 2. the overall oxidative activity of mitochondria within a tissue, and 3. total tissue mass. Perturbations in BCAA oxidation have been implicated in many disease contexts, underscoring the importance of BCAA homeostasis in overall health.